Stem cells are undifferentiated cells that can differentiate into various types of cells constituting biological tissues and can be obtained from the tissues of embryos, fetuses and adults. Among the different cell types of the stem cell, pluripotent stem cells refer to the stem cells that can differentiate into any of the three germ layers, i.e., the endoderm, mesoderm and ectoderm.
The stem cells can be classified based on their anatomical sites, cellular functions, antigens presented on the cell surface, transcription factors, proteins produced by the cells, and specific cell types that can be derived from the stem cells.
As a rather clear criterion of classification, the stem cells can be classified based on their origin. Embryonic stem cells (ES cells) are isolated from embryos and adult stem cells are isolated from adult tissues.
Alternatively, the stem cells can be classified into pluripotent, multipotent and unipotent stem cells based on their capacity to differentiate into specialized cell types. In general, embryonic stem cells (ES cells) can be classified as pluripotent stem cells and adult stem cells can be classified as multipotent and unipotent stem cells.
The embryonic stem cells (ES cells) derived from the inner cell mass of a blastocyst, an early-stage embryo, are pluripotent stem cells that can differentiate into all the tissues constituting the adult body. That is to say, the embryonic stem cells are undifferentiated cells that can proliferate without limit and can differentiate into all cell types. Unlike the adult stem cells, they can be inherited to the next generation because they can form germ cells.
However, the pluripotent embryonic stem cells raise serious religious and ethical concerns implicated with the destruction of embryos during preparation thereof. In addition, since they are derived from limited embryos, immune rejection due to lack of immunocompatability between individuals cannot be avoided. To overcome these problems, there have been various attempts to artificially prepare pluripotent stem cells such as induced embryonic stem cells or embryonic stem cells using the cells derived from adults.
Typical examples include somatic cell nuclear transfer (SCNT), fusion with ES cells and reprogramming by defined factors. The somatic cell nuclear transfer is very inefficient and there is an ethical question in that it requires eggs in large quantities. The fusion with ES cells has a serious problem in terms of cell stability because the induced cells additionally have two pairs of genes. The reprogramming by defined factors, which has been reported most recently, involves the serious problem of carcinogenesis because it uses oncogene-containing viruses.
Therefore, a method for preparing induced pluripotent stem cells with proven stability and safety without raising ethical problems is needed for the development of a cell therapy agent.
To satisfy this need, a method for inducing induced pluripotent stem cells through dedifferentiation by introducing four genes into somatic cells was studied (Takahashi K, Yamanaka S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676). This method is free from ethical concerns because adult cells are used and the immune rejection problem is solved because autologous cells are used.
The inventors of the present disclosure have acquired dedifferentiated stem cells from an extract of animal-derived induced pluripotent stem cells (iPS) but there are some limitations.
First, a large quantity (20 mg or more) of iPSC extract is necessary for this method. For this reason, induction of dedifferentiation using an extract of human-derived dedifferentiated stem cells, which is costly and requires much labor, has not been successful. For example, to prepare human-derived dedifferentiated stem cells for obtaining 20 mg of extract, an expert has to work hard for at least 3 months, which is very costly.
Second, when somatic cells to be induced are treated with an extract of animal stem cells or dedifferentiated stem cells thereof, if the cells survive in the extract without being completely destroyed, it is not easy to distinguish the dedifferentiation-induced cells from the surviving dedifferentiated stem cells and analysis of genomic DNA is necessary, which is costly and time-consuming.
Third, since preparation of human-derived dedifferentiated stem cells using proteins has been hardly successful, an extract of human-derived dedifferentiated stem cells prepared using viruses has to be used. Because the resulting cells may contain oncogenic substances derived from the viruses, there may be difficulty in clinical application.